PROJECT SUMMARY/ABSTRACT Non-alcoholic steatohepatitis (NASH) is an emerging epidemic of liver disease in the US and the basis for a rising incidence of hepatocellular carcinoma. NASH-associated fibrosis, regardless of other histologic features such as inflammation, is the major predictor of long-term outcomes in patients. Accordingly, there are increas- ing numbers of clinical drug trials to slow down or reverse fibrosis progression in patients with NASH. However, no drugs have been approved yet for widespread use. The direct fibrogenic mediators of liver fibrosis are he- patic stellate cells (HSCs), which become activated/differentiate into myofibroblasts that deposit excessive ex- tracellular matrix (ECM) proteins in an aberrant wound healing cascade. The stiff matrix produced by activated HSCs leads to the loss of major functions in hepatocytes. The differentiation of HSCs into myofibroblasts and their interactions with hepatocytes in NASH is the result of the complex crosstalk between numerous microen- vironmental signals. Thus, treating NASH-associated fibrosis effectively will require understanding and inter- rupting this complex crosstalk that distorts liver architecture and leads to liver decompensation. Differences across species in drug metabolism and disease pathways necessitate supplementation of animal data with human-relevant in vitro assays. Despite important progress in the development of culture techniques to stabilize the phenotype of primary human hepatocytes (PHHs) in culture for several weeks, there is a need to develop a platform that enables the investigation of PHH-HSC interactions within physiological and disease settings. We have developed a cellular microarray that allows simultaneous modulation of the size/composition of patterned ECM protein domains, substrate stiffness, and soluble factor concentrations, while also enabling parallel measurements of cellular phenotype and contractility. Here, we will adapt this cellular microarray to test our hypothesis that the ECM protein composition, substrate stiffness, and soluble factors act collectively to modulate the phenotypes of PHHs and HSCs and their interactions in an NASH-like microenvironment. Our approach will enable hypothesis-driven studies incorporating controlled perturbations of extracellular signals. In aim 1, we will examine the effects of ECM composition and substrate stiffness on long-term phenotypic re- sponses of PHHs under normal and NASH-inducing conditions. In aim 2, we will investigate the cooperative microenvironmental regulation of the activation states of primary human HSCs. In aim 3, we will develop a co- culture approach to determine the roles of reciprocal interactions between PHHs and HSCs and establish a platform for evaluating NASH-relevant therapeutics. Our studies will reveal mechanisms underlying phenotypic alterations of human HSCs and PHHs, including interconnections between biochemical and biomechanical sig- nals. These efforts will aid the development of drugs aimed at reversing fibrosis.